JPH07278130A - Physiologically active substance ei-1507s - Google Patents

Abstract

PURPOSE:To obtain new physiologically active substance EI-1507s, having antimicrobial and inhibiting actions on the production of interleukin-1 and useful as a therapeutic agent for chronic articular rheumatism, gout, osteoarthritis, osteoporosis, periarteritis nodosa, colitis ulcerosa, etc. CONSTITUTION:The physiologically active substance EI-1507s are expressed by the formula (X and Y together are 0 or independently H or OH), e.g. the compound EI-1507-1 in which X and Y together are 0 and the compound EI-1507-7-2 in which X and Y each is H or OH. The compounds expressed by the formula are obtained by culturing a microorganism belonging to the genus Streptomyces. The substance EI-1507s expressed by the formula are useful as a therapeutic agent for symptoms such as chronic nephritis, active chronic hepatitis, sepsis, endotoxic shock, atherosclerosis and pyrexia related to infectious diseases, systemic scleroderma, etc.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a physiologically active substance EI- having an antibacterial action and an interleukin-1 production inhibitory action.
Regarding 1507 class. Interleukin-1 is known to be involved in various inflammatory diseases. Therefore E
Class I-1507 is used for rheumatoid arthritis, gout, osteoarthritis, osteoporosis, periarteritis nodosa, ulcerative colitis, chronic nephritis, active chronic hepatitis, sepsis, endotoxic shock, atherosclerosis, and infectious diseases. It is useful as a therapeutic agent for symptoms such as fever and systemic scleroderma.

[0002]

2. Description of the Related Art Interleukin-1 (hereinafter simply referred to as IL-
1) are various cells in the body, such as macrophages and monocytes, neutrophils, fibroblasts, skin keratinocytes, liver Kuffer cells, renal glomerular mesangial cells, brain astrocytes, glial cells, It is a protein with a molecular weight of 17.5 kDa produced from vascular endothelial cells and has an α type with an isoelectric point (pI) of 5 and a β type with a pI of 7. Currently α and β
The mold is known to have the same effect. IL-1 is known to have a wide variety of biological activities. That is, IL-1 is said to act as a factor that enhances the reactivity of lymphocytes to mitotic proliferation and as a cofactor that enhances B cell proliferation and antibody production. Further, IL-1 is considered to be involved in fever by acting on the arachidonic acid cascade in the temperature center of the hypothalamus and enhancing prostaglandin E ₂ synthesis. Furthermore, IL-1 has been shown to have a significantly increased activity in the serum of patients with sepsis, Coulomb's disease, etc., and in the joint cavities of patients with rheumatoid arthritis, and IL-1 for the onset and progression of these diseases. 1
Has been suggested to be involved. It is considered that suppression of IL-1 production is effective in alleviating such IL-1-mediated symptoms. As a substance having an IL-1 production suppressing action, a naphthalene derivative (Japanese Patent Laid-Open No. 4-593743) and a 3-arylisothiazole derivative (Japanese Patent Laid-Open No. 4-59474) have hitherto been used.
No. 74121), and a gingerol derivative (Japanese Patent Laid-Open No. Hei 4-
Synthetic compounds such as 202127) are known, but EI-1507s provided by the present invention are obviously different from these.

[0003]

An object of the present invention is to provide a physiologically active substance having an antibacterial action and an IL-1 production inhibiting action.

[0004]

[Means for Solving the Problems] With the aim of providing useful physiologically active substances, as a result of research on many microbial products obtained from the natural world, microbial cultures isolated from soil It was found that a physiologically active substance having an IL-1 production inhibitory action is produced therein. Then, the substance was isolated and purified, and its physicochemical properties were examined. As a result, it was found to be a novel substance, and the substance was named EI-1507.

According to the invention, the general formula (I)

[0006]

[Chemical 2]

## STR1 ## wherein X and Y together represent an oxygen atom, or independently a hydrogen atom or a hydroxyl group, and a physiologically active substance EI-1507 can be provided. . The substance is Streptomyces ( S
It can be obtained by culturing a microorganism belonging to the genus treptomyces ). The present invention will be described in detail below.

Compound EI-15 represented by the general formula (I)
In group 07, a compound in which X and Y are together an oxygen atom is EI-1507-1, and a compound in which X and Y are independently a hydrogen atom or a hydroxyl group is EI-.
1507-2. The physicochemical properties of EI-1507 are as shown below.

(I) EI-1507-1 Properties: Light brown amorphous powder Melting point: 177 to 178 ° C. Molecular weight: 354 Molecular formula: C ₂₀ H ₁₈ O ₆ High resolution mass spectrometry: (Matrix: m- Nitrobenzyl alcohol): m /
z Actual value 355.1198 Calculated value 355.1181 [C ₂₀ H ₁₈ O ₆ + H] ⁺・ Infrared absorption spectrum (KBr method): ν _max (cm ^-1 ): 3421,1685,1672,1300,1264,1026,752・ Ultraviolet absorption spectrum (in methanol): λ _max (nm): 300 (ε = 7,400), 256 (ε = 8,200), 215 (ε =
24,700) ・¹ H-NMR spectrum (500MHz, CDCl ₃ solution): δ ppm
(Multiplicity, coupling constant, integral): 7.46 (t, J = 7.8Hz, 1H), 7.4
1 (dd, J = 1.2,7.8Hz, 1H), 7.12 (dd, J = 1.2,7.8Hz, 1H), 6.99
(d, J = 9.6Hz, 1H), 6.48 (d, J = 9.6Hz, 1H), 5.91 (d, J = 5.1Hz, 1
H), 3.94 (s, 3H), 3.00, (d, J = 16.3Hz, 1H), 2.80 (d, J = 17.8H
z, 1H), 2.73 (d, J = 17.8Hz, 1H), 2.60 (d, J = 16.3Hz, 1H), 2.24
(d, J = 5.1Hz, 1H), 2.11 (s, 1H), 1.38 (s, 3H) ・¹³ C-NMR spectrum (125MHz, CDCl ₃ solution): δ ppm
(Multiplicity): 193.1 (s), 192.8 (s), 156.7 (s), 146.6 (s), 13
3.2 (s), 132.8 (s), 132.2 (d), 130.6 (d), 127.7 (s), 127.2
(s), 119.4 (d), 114.9 (d), 70.7 (s), 65.7 (s), 63.6 (d), 61.3
(s), 56.0 (q), 51.8 (t), 44.4 (t), 28.5 (q), ・ Specific rotation: [α] _D ²⁴ = +435.7 ° (c = 0.05, CHCl ₃ ) ・ Solubility: Easily soluble in methanol and acetone, ethyl acetate,
Soluble in chloroform, water ・ Color reaction: Positive for iodine, sulfuric acid, anisaldehyde ・ Thin layer chromatography: Rf value: 0.62 Developing solvent: Chloroform-methanol 9: 1 Thin layer: Kieselgel 60F ₂₅₄ (Merck, Art.5628) Development method: room temperature, rising method, 20-40 minutes Detection: 253.6 nm ultraviolet irradiation method

(Ii) EI-1507-2 ・ Property: Light brown amorphous powder ・ Melting point: 192-194 ° C. ・ Molecular weight: 356 ・ Molecular formula: C ₂₀ H ₂₀ O ₆・ High resolution mass spectrometry: (Matrix: m- Nitrobenzyl alcohol): m /
z Actual value 339.1231 Calculated value 339.1232 [C ₂₀ H ₂₀ O ₆ -H ₂ O + H] ⁺・ Infrared absorption spectrum: (KBr method): ν _max (cm ^-1 ): 3357,1670,1592,1473, 1315,1261,752 ・ Ultraviolet absorption spectrum (in methanol): λ _max (nm): 300 (ε = 7,500), 283 (ε = 9,600), 220 (ε =
15,100,) ・¹ H-NMR spectrum (500MHz, CDCl ₃ solution): δ ppm
(Multiplicity, coupling constant, integral): 7.36 (dd, J = 7.7,8.1Hz, 1
H), 7.19 (d, J = 9.5Hz, 1H), 7.02 (dd, J = 0.8,7.7Hz, 1H), 6.93
(dd, J = 0.8,8.1Hz, 1H), 6.51 (d, J = 9.5Hz, 1H), 6.14 (s, 1H),
5.74 (d, J = 9.4Hz, 1H), 3.90 (s, 1H), 2.96 (d, J = 18.0Hz, 1H),
2.8 (d, J = 18.0Hz, 1H), 2.78 (m, 2H), 2.46 (d, J = 9.4Hz, 1H),
1.77 (s, 1H), 1.42 (s, 3H) ・¹³ C-NMR spectrum (125MHz, CDCl ₃ solution): δ ppm
(Multiplicity): 200.5 (s), 157.3 (s), 150.7 (s), 138.8 (d), 13
6.0 (s), 131.4 (d), 130.4 (d), 128.9 (s), 122.9 (d), 122.8
(s), 111.2 (d), 72.0 (s), 69.9 (d), 63.9 (s), 63.6 (d), 63.1
(s), 55.8 (q), 53.4 (t), 45.8 (t), 29.4 (q) ・ Specific rotation: [α] _D ²⁴ = + 33.3 ° (c = 0.07, CHCl ₃ ) ・ Solubility: Soluble in methanol, acetone, ethyl acetate, chloroform, water ・ Color reaction: Positive for iodine, sulfuric acid, anisaldehyde ・ Thin layer chromatography: Rf value: 0.59 Developing solvent: Chloroform-methanol 9: 1 Thin layer: Kieselgel 60F ₂₅₄ (Merck, Art.5628) Development method: room temperature, rising method, 20 to 40 minutes Detection: 253.6 nm ultraviolet irradiation method From the above data, EI-1507-1 and EI-15
07-2 was found to be a new compound.

The above data was measured by the following equipment.・ Melting point: Yanagimoto Corporation Micro melting point measuring device ・ Specific rotation: JASCO DIP-370 polarimeter ・ Mass spectrometry: JEOL HX110A mass spectrometer ・ Infrared absorption spectrum: JEOL JIR-RFX3001 infrared absorption spectrometer ・ Ultraviolet Absorption spectrum: Shimadzu UV-2200 type ultraviolet absorption spectrometer NMR spectrum: Bruker AM500 type nuclear magnetic resonance spectrometer Next, the pharmacological activity of compound EI-1507 will be described in Test Examples 1 and 2.

Test Example 1: Antibacterial activity Antibacterial activity was 3 g of Bactrypton (manufactured by Difco).
/ Liter, meat extract 3g / liter, yeast extract 1
Medium (pH 7.0) consisting of g / liter, glucose 1 g / liter and agar 16 g / liter
Was measured by the agar dilution method.

Minimum inhibitory concentration (MI) against various bacteria
C) is shown in Table 1.

[0014]

[Table 1]

Test Example 2 Inhibitory effect on IL-1 production Compounds EI-1507 of the present invention I from human monocyte-derived cell line THP-1 cells (ATCC No. TIB 202)
The L-1β production inhibitory effect was examined. IL-1β amount is EL
It was measured by the ISA method.

1 × of THP-1 cells was added to RPMI1640 (manufactured by Nissui) culture medium containing 10% inactivated fetal bovine serum.
The cells were suspended and suspended at a concentration of 10 ⁵ cells / ml. This was dispensed into a 24-well plate at a volume of 1 ml / well, and phorbol 12-millistart 13-acetate (PMA: final concentration; 30
nM) was added, and the cells were cultured at 37 ° C., 5% CO ₂ /95% air for 65 hours to differentiate the cells into macrophage-like.

The culture plate was gently washed with serum-free RPMI 1640 to remove non-adherent cells, and then serum-free RPMI 1640 to which lipopolysaccharide (LPS: final concentration; 25 μg / ml) and a test sample were simultaneously added. The cells were cultured for 4 hours in the culture solution (1 ml / well). After the culture, the amount of IL-1β released in the culture supernatant was measured using an IL-1β measurement kit (Amersham).

The IL-1 production inhibition rate was calculated by the following equation:
C ₅₀ (50% inhibitory concentration) was determined.

[0019]

[Equation 1]

Results 50% of IL-1 production of THP-1 cells by LPS stimulation
The inhibitory concentration was 1.2 μM for EI-1507-1, EI150
7-2 was 1.5 μM. From this result, the EI of the present invention
It is clear that -1507s have an IL-1 production inhibitory effect. Next, a method for producing EI-1507 will be described.

EI-1507s belong to the genus Streptomyces, microorganisms capable of producing EI-1507s are cultured in a medium, and EI-150 is added to the culture.
It can be obtained by producing and accumulating 7 groups and collecting EI-1507 from the culture. EI-150
As a 7-class producing microorganism, it belongs to the genus Streptomyces,
Any strain can be used as long as it has the ability to produce EI-1507s. In addition, any strain obtained by mutating these strains by an artificial mutagenesis method, for example, ultraviolet irradiation, X-ray irradiation, treatment with a mutagen, or a naturally mutated strain may have the ability to produce EI-1507s. For example, it can be used in the present invention. As specific preferred examples, the present inventors have actinomycete Streptomyces sp was isolated from the soil of Yamagata Prefecture (Streptomyces
sp.) E-1507 strain.

[0022] Streptomyces sp (Strep
The mycological properties of the tomyces sp.) E-1507 strain are as follows. 1. Morphological properties

[0023]

[Table 2]

2. Culture properties E-1507 strain shows normal or vigorous growth in commonly used synthetic and natural media, and the basal hyphae show a yellow to brown system. A brownish soluble pigment may be produced by the medium. 28 ℃ on various media, 14
The characteristics of growth and color when cultured for one day are shown below. The colors are displayed in the Color Harmony Manual (Container Cor
according to the color classification by the poration of America).

[0025]

[Table 3]

3. Physiological Properties Physiological properties of the E-1507 strain are shown below. The growth temperature range is 10 days after culturing, and the other results are at 28 ° C. after culturing for 2-3 weeks. -Growth temperature range: 8.0-40.0 ° C-Liquefaction of gelatin; None-Hydrolysis of starch; Yes-Coagulation and peptization of skim milk powder; Peptone conversion. -Production of melanin-like pigment (1) Peptone-Yeast-Iron agar medium; Yes (2) Tyrosine agar medium; Yes-Availability of carbon source Predham-Gottlieb agar medium was used as the basal medium. Hereinafter, + means to use, and − means not to use. L-arabinose; + D-xylose; + D-glucose; + sucrose; + raffinose; -D-fructose; + rhamnose; + inositol; + D-mannitol; +

4. Chemotaxonomic properties-Optical isomers of diaminopimelic acid in bacterial cells; LL type Based on the mycological properties above, this strain is classified into Streptomyces genus among actinomycetes. Therefore, this strain was designated as Streptomyces sp. E-1.
Named 507, the Institute of Biotechnology, Industrial Technology Institute
Deposited as FERM BP-4623 on March 30, 1994.

For culturing the EI-1507-producing bacterium of the present invention, a usual culturing method used for culturing a microorganism of the genus Streptomyces is applied. The medium used may be either a natural medium or a synthetic medium as long as it contains a carbon source, a nitrogen source, an inorganic substance and the like that can be assimilated by the bacterium. Carbon sources include glucose, fructose, sucrose, stabilose, starch, dextrin, carbohydrates such as mannose, maltose, molasses, organic acids such as citric acid, malic acid, acetic acid, fumaric acid, alcohols such as methanol and ethanol, methane. Hydrocarbons such as ethane, propane and n-paraffin, amino acids such as glutamic acid and glycerol are used.

As the nitrogen source, ammonium salts such as ammonium chloride, ammonium sulfate, ammonium nitrate and ammonium phosphate, amino acids such as aspartic acid, glutamine, cystine and alanine, urea, peptone, meat extract, yeast extract, dry yeast and corn steep.・ Liquor, soybean powder, cottonseed meal, soybean casein, casamino acid, Pharmamedia, etc. are used.

Inorganic substances include potassium monohydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, copper sulfate, cobalt sulfate, zinc sulfate, calcium pantothenate, molybdic acid. Ammonium, potassium aluminum sulfate, barium carbonate, calcium carbonate, cobalt chloride, salt and the like are used.

In addition, if necessary, the growth of microbial cells such as vitamins such as thiamine or EI-1507 is added to the medium.
It is possible to add substances that promote the production of the class. If the microorganism used requires a specific substance, it is necessary to add what is necessary for growth. Culture is shaking culture method,
It is carried out at a temperature of 20 to 40 ° C. and a pH in the vicinity of neutral by aeration stirring culture method or the like.

EI-150 is usually obtained by culturing for 3 to 7 days.
The accumulation of the 7th species reaches the maximum, and the culture is completed. When the EI-1507s accumulated in the culture are isolated and collected from the culture, a usual method for collecting a physiologically active substance from the culture is applied. That is, acetone, extraction of cell components with a solvent such as methanol, filtration, cell removal by centrifugation, adsorption resin, silica gel, silanized silica gel, reverse phase silica gel, aluminum, cellulose, diatomaceous earth, magnesium silicate, The EI-1507s are isolated by adsorption / desorption treatment of the active substance by column chromatography or thin layer chromatography using a gel filtration agent, an ion exchange resin or the like, partitioning with an appropriate solvent system and the like.

The detection of EI-1507s in the above purification step can be carried out by silica gel thin layer chromatography, followed by iodine color development, or by a 253.6 nm ultraviolet irradiation method. Examples of the present invention will be shown below.

[0034]

【Example】

Example 1 Streptomyces species E-1 as an inoculum
507 strain, glucose 10g as the first type medium
/ Liter, soluble starch 10 g / liter, beef extract (Kyokuto Pharmaceutical Co., Ltd.) 3 g / liter,
Powdered yeast extract S (Nippon Pharmaceutical Co., Ltd.) 5 g / liter, bactotryptone 5 g / liter, potassium dihydrogen phosphate 1 g / liter, magnesium sulfate heptahydrate 0.5
g / l and magnesium phosphate octahydrate.
A medium (pH 7.0) having a composition of 5 g / liter was used. 1 platinum loop of the inoculum was inoculated into 10 ml of the type 1 medium contained in a 50 ml thick type test tube, and 5 test tubes (total medium amount 5
0 ml) was shake-cultured at 28 ° C. for 3 days.

40 ml of the first culture broth was inoculated into 5 ml of 45 ml of the second culture medium in a 300 ml Erlenmeyer flask (total 8 flasks, 400 ml). The composition of the second type medium is the same as that of the first type medium. The second seed culture was performed by shaking culture at 28 ° C. for 2 days. 300 ml of the obtained second seed culture solution was placed in a 2 liter Erlenmeyer flask and 350 ml of the third seed medium.
50 ml each (total 6 flasks, 2.4 liters)
Inoculated. The composition of the third-type medium is the same as that of the second-type medium. The third seed culture was performed by shaking culture at 28 ° C. for 2 days.

1.8 liter of the obtained third-type culture solution was added to 3
Inoculation was performed on 17 liters of the main fermentation medium in a 0 liter stainless steel jar fermenter. As a main fermentation medium, HP-20 10% (V / V), soluble starch 40
g / liter, soybean flour 10 g / liter, corn steep liquor 5 g / liter, dry yeast 5 g / liter, potassium dihydrogen phosphate 0.5 g / liter,
Magnesium phosphate octahydrate 0.5 g / liter, zinc sulfate heptahydrate 10 μg / liter, cobalt chloride 6
Hydrate 1 μg / liter and nickel sulfate 1 μg
A medium (pH 7.0) having a composition of 1 / liter was used. This main fermentation culture was performed at 28 ° C. for 4 days by aeration and agitation culture (rotation speed: 250 rpm, aeration amount: 18 liters / minute).

17 liters of the obtained main fermentation culture broth was centrifugally filtered to separate cells and HP-20, and the culture supernatant 14
Got liters. This was passed through a Diaion HP-20 column (2 liters) for adsorption. 30% column
After washing with 10 liters of methanol, it was eluted with a solvent of methanol: acetone = 7: 3. Fractions containing EI-1507 were collected, concentrated to dryness under reduced pressure, and then dissolved in ethyl acetate. 2 liter of the obtained solution was mixed well with the same amount of water,
After partitioning, the ethyl acetate layer was collected using a separating funnel.
The aqueous layer was again mixed well with the same amount of ethyl acetate and partitioned, and the ethyl acetate layer was recovered using a separating funnel. The respective ethyl acetate layers were combined and concentrated to dryness under reduced pressure to obtain 4.5 g of a brown solid. This was dissolved in chloroform and passed through a silica gel column (500 ml) for adsorption, and then the chloroform: methanol ratio was 99: 1, 9.
Elution was carried out successively with 2 liters of each solvent, which was 3: 7 and 90:10. Fractions containing EI-1507s were collected and concentrated to dryness under reduced pressure to obtain 3.9 g of brown powder. The obtained powder was dissolved in 50% methanol and the ODS column (ODS-
A 60-230 / 70; made by YMC: 2.5 liter) and passed through a column and eluted with a linear concentration gradient of methanol (50 to 70% / 15 hours). Fractions containing EI-1507s were concentrated to dryness under reduced pressure and dissolved in 50% methanol. This is again subjected to ODS column fractionation under the following conditions, and EI-150
7-1 (320 mg), EI-1507-2 (340 m
g) was obtained.

The EI-1507s in the above process were detected by silica gel thin layer chromatography followed by iodine color development, or by the 253.6 nm ultraviolet irradiation method. ODS column preparative conditions Column: ODSAQ120-S50 (manufactured by YMC) φ2
0 × 500 mm Eluent: 50% methanol Flow rate: 10 ml / min

[0039]

According to the present invention, antibacterial action and IL-
It is possible to provide EI-1507, which is a physiologically active substance having an inhibitory action on 1 production.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication location A61K 31/34 ABX ACR ACS ACV ADC ADT ADZ C12P 17/02 7432-4B (C12P 17/02 C12R 1 : 465) (72) Inventor Sadao Teziba 1626-9 Nogaya-cho, Machida-shi, Tokyo (72) Inventor Tsutomu Azuma 3-6-6 Asahi-cho, Machida-shi, Tokyo (72) Mayumi Yoshida Sagamihara, Kanagawa 9-18 Isobe, Japan (72) Inventor, Yuzuru Matsuda 1-22-7 Nukiinami-cho, Koganei-shi, Tokyo

Claims (1)

[Claims] 1. A compound represented by the general formula (I): (In the formula, X and Y together represent an oxygen atom or independently a hydrogen atom or a hydroxyl group.)
A physiologically active substance EI-1507 represented by.